Field of the Invention
A polypeptide esterase that is resistant to inactivation by heavy metals and other extreme conditions. Polynucleotides encoding this esterase.
Description of the Related Art
Until lately, and despite its uniqueness, the Red Sea has received little attention among marine environments. The Red Sea formed 3-5 million years ago when the Arabian and African plates started to split1. It is characterized by high temperature and salinity owing to the high rate of evaporation, lack of major river inflows and a low rate of rainfall1. The Red Sea is characterized by the presence of deep-sea hypersaline anoxic basins; called brine pools, which are large bodies of water at the bottom of the ocean characterized by high temperature and salinity. To date, twenty-five brine pools have been found in the Red Sea1, 2. Atlantis II Deep (FIG. 1) is the largest brine pool in the Red Sea, has the highest temperature and is the most dynamic1,3. It has a maximum depth of 2,194 m and is stratified into several layers that increase in temperature and salinity with increasing depth; the brine-seawater interface, upper convective, middle convective and lower convective layers (LCL)1, 3. The lowest layer; LCL is characterized by a temperature of 68.2° C., pH value of 5.3 and salinity of 270 psu, which is 7.5 times that of normal seawater1, 3. Atlantis II Deep is nearly anoxic and has high concentrations of iron, zinc, copper and other heavy metals1, 3. Together, these extreme conditions make the Atlantis II brine pool an attractive site for mining for biocatalysts, such as lipolytic enzymes, which are predicted to possess desirable traits, including and not limited to, thermo-tolerance, halo-tolerance, pH plasticity and resistance to inhibition by heavy metals.
Industrialized societies are moving towards white (industrial) biotechnology, which has proven to be environmentally sound and commercially efficient4. This poses a continuous demand for novel biocatalysts, preferably biocatalysts that demonstrate high activity over a wide range of conditions such as temperature, salinity, pH and metal concentration. Biocatalysts of microbial origin represent the majority of biocatalysts used in industrial and biotechnological processes5. This owes to the capability of prokaryotes to populate and adapt to different environments, from hydrothermal vents to Antarctic desert soil, from which a wide array of biocatalysts are derived that are robust within a flexible range of conditions; making them desirable for industry6.
Metagenomics serves as a powerful tool to access the genomes of the unculturable majority of prokaryotes, and to investigate their potential as sources of novel biocatalysts. It has led to the identification and characterization of a vast number of biocatalysts that are active under a wide range of conditions reflecting the environment from which they originate, making them desirable for industrial use7-10.
Microbial lipolytic enzymes possess a huge potential as industrial biocatalysts. They are characterized by substrate specificity, regio- and enantioselectivity that surpasses that of any other enzyme, making their application potential boundless11. Using lipolytic enzymes in industrial and biotechnological applications is estimated to be a billion dollar business12. Their applications include and are not limited to leather manufacture, flavor development in the dairy industry, oil biodegradation and the synthesis of pharmaceuticals and chemicals12-15.
As of 2005, only a dozen thermostable lipases/esterases had been isolated; Rhee J-K et. Al. (2005)45, New thermophilic and thermostable esterase with sequence homology to the hormone sensitive lipase family, cloned from a metagenomic library. Appl Environ Microbiol Vol. 71(2): pp. 817-825. A 2010 paper reported that, surprisingly, only 7 esterases of thermophilic origin had been sequenced. Yu, et al. (2010)46, Gene cloning and characterization of a novel thermophilic esterase from Fervidobacterium nodosum Rt17-B1, Acta Biochim. Biophys. Sin., Vol. 42(4), pp. 288-295 described a new candidate termed FNE acetylesterase, isolated from Fervidobacterium nodosum strain Rt17-B12. Another publication, Waters D M et al (2012)47, Cloning, Overexpression in Escherichia coli, and Characterization of a Thermostable Fungal Acetylxylan Esterase from Talaromyces emersonii, Appl. Environ. Microbiol. Vol. 78(10): pp. 3759-3762 recently identified thermostable esterase from Talaromyces emersonii bears sequence homology to acetylxylan esterases.
The global market for lipases is significant. The division of the entire market for lipases is detergent (42%), pulp and paper (about 7%), leather (about 6%), dairy products (about 17%), and sweeteners (about 21%) (otd.unc.edu/documents/11_4_2010_Williams.pptx). The biofuels market, which is expected to grow significantly in its need for novel biocatalysts, is seen as the greatest opportunity for expanding the use of esterases. Over 300 industrial processes have been designed that rely on biocatalysts (Singh R K et al (2013)48, From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes. Int. J. Mol. Sci. Vol. 14: pp. 1232-1277). Esterases are of particular use in the production of bulk chemicals and pharmaceuticals, where they find very specific niches in chemical production. Examples include precursors for pyrethrin insecticides; in the production of naproxen; solubilization of certain antibiotics; and often as a general mild remover of protective groups on chemical intermediates during various syntheses (Bornscheuer UT (2002)49 Microbial carboxyl esterases: classification, properties, and application in biocatalysis. FEMS Microbiol. Rev. Vol. 26: pp. 73-81). A recent paper described the use of a thermostable esterase from Archaeoglobus fulgidus (Cao H et al (2012)50 Biocatalytic synthesis of poly (δ-valerolactone) using a thermophilic esterase from Archaeoglobus fulgidus as catalyst. Int. J. Mol. Sci. Vol. 13: pp 12232-12241) for producing polymers useful in preparing nanoparticles for targeted therapeutic delivery, as an example. Esterases that have recently received particular attention in industrial use include furoyl esterases, pectin esterases, acetylxylan esterases, and rhamnogalacturonan acetyl esterases. The first two types are commonly used in food processing, while the latter two find use in biomass solubilization. In addition to the biofuels market, enzymatic cleavage of these molecules can contribute to production of components of nutraceuticals, cosmetics, and fine chemicals.